Revision as of 08:32, 6 April 2010

Contents

Overview

The following is a procedure for the in vitro modification of DNA before electrotransformation into Lactobacillus plantarum developed by Alegre et al. The inability to recover successful transformants in many lactic acid bacteria including Lactobacillus plantarum is most likely the result of active host restriction mechanisms. This method was originally developed for Saccharopolyspora spinosa in an attempt to circumvent the active restriction-modification of the host bacterium.

Procedure

Preparation of the Extract

1. Prepare a stock solution of AEBSF in water at 0.2 mM and store in a 4°C fridge.
2. Grow up 45 ml of L. plantarum cells in MRS overnight and wait until OD600 is between 1.5 and 2.0.
3. Pellet cells at maximum speed until supernatant is clear (∼4 mins @ 5000g).
4. Resuspend pellet in 10 ml of wash buffer and centrifuge again.
5. Resuspend in 2 ml of wash buffer and put cells on ice.